Method of storing blood and a blood storage bag therefore

ABSTRACT

A method of storing blood is disclosed in which a plastic blood collection bag is further supplied with silicone rubber into which pure Ca(OH) 2  has been compounded. The method is an efficient way to prolong the useful life of blood, especially the erythrocytes, since their viability can be enhanced by removal of CO 2  from the blood. Such removal can be a method of maintaining an adequate pH balance in the complex blood system which appears to be essential to maintaining the blood in storage for prolonged periods of time.

This is a division, of application Ser. No. 821,668, filed Aug. 4, 1977now U.S. Pat. No. 4,162,676.

BACKGROUND OF THE INVENTION

The anatomy and physiology of blood has been of great interest to thehuman race for a long time, because, as recognized centuries ago, theblood system is the fluid pipeline that maintains the complex chemicalbalance of the human body. The blood system carries nutrients to theother living tissues of the body and at the same time carries away thewaste by-products of the multitudinous complex chemical reactions thatare going on inside the body and which are essential to life itself.

Severe intrusions on body tissues and/or the blood system itself oftenrequires the supplementation of blood to the blood system. Generally, ifthe intrusion is not too severe or the loss of blood is not too copious,the body has a unique system for supplementing or replacing lost blood.

In those instances when the body cannot replace or supplement the neededblood because of sheer volume loss or because the body has aphysiological malfunction, the replacement or supplementation can comefrom an external source, such as stored blood.

Historically, the storage of blood outside the body is not very old.With the onset of the Second World War, the need for large volumes ofreplacement blood brought on a flurry of activity in researching thebest methods of storing blood.

Since the end of the Second World War and up to approximately thebeginning of the present decade, the systems for storing blood that weredeveloped were those used during the war period. At the very best,however, the duration of storage of whole blood in its liquid form, was21 days.

Such storage systems usually involved the storage of blood in an AcidCitrate-Dextrose solution.

Recently, Dr. Lee Wood and Dr. Ernest Beutler in a publication,Transfusion, Vol. 11, No. 3, May-June, 1971, pp. 123-133, reported thatblood could be stored for 35 days by the co-use of adenine with AcidCitrate-Dextrose solutions and that such a practice was the primarystorage system in Sweden.

They further set forth a method they had developed for the storage oferythrocytes. They remove the plasma from whole blood and store theerythrocytes in an artificial media. Their work shows that they can getequivalent storage to the ACD-adenine storage system.

One of the major problems in the storage of whole blood is that theerythrocytes produce large quantities of lactic acid from glucose. Thisphenomena proceeds even when the blood is stored at low temperatures.The presence of the lactic acid, among other things, contributes to thecontinued decrease in the pH of the stored whole blood.

The lowering of the blood pH has a dramatic effect on the viability ofthe erythrocytes when the blood is transfused.

The mechanisms involved in the effect are described by Wood and Beutlerin an article entitled "Preservation of Red Cell 2,3-DPG and Viabilityin Bicarbonate-Containing Medium: The Effect of Blood Bag Permeability",Journal of Lab and Clinical Medicine, Vol. 80, No. 5, p.p. 723-728, "Thefall in the pH of the stored cells results initially in the loss oftheir 2,3-diphosphoglycerate (2,3-DPG), so necessary for efficientdelivery of oxygen to the tissues. Eventually, glycolysis is choked offat the pH-sensitive hexokinase and phosphofructokinase steps, so thatthe erythrocyte loses its capacity to live and circulate whentransfused. Thus, regulation of pH plays a key role in liquidpreservation".

Wood and Beutler go on to state the essential problem, "the use of veryalkaline blood preservative solutions is unsatisfactory, however. Veryhigh pH levels result in reduction of NAD to NADH in thelactate-pyrovate system, and thus impede glycolysis at theglyceraldehyde phosphate dehydrogenose step. Although alkalinepreservatives result in good 2,3-DPG maintenance, adenosinetriphosphate(ATP) is rapidly depleted under these conditions, and viability is poor.To some extent, this effect may be counteracted by the addition ofpyrovate to reoxidize NADH, but even very alkaline preservatives cannotabsorb enough hydrogen ions to maintain the pH level above the criticalvalues required to prevent the decomposition of 2,3-DPG.

Clearly, it would be helpful to have a highly efficient buffer systemwhich would maintain the pH of preserved cells above 7.4 in the face ofthe production of large amounts of lactic acid. Yet, no buffer ion whichcan absorb this large amount of acid and yet be reinfused is known".

Prior art attempts at preventing degeneration of whole blood sufferingfrom the disadvantages described above, that is, generally the buffersolutions of sufficient strength required to maintain the high pH levelhave the disadvantage of not being compatible enough to reinfuse in thehuman without deleterious side effects. Further, attempts have been madeto include small pouches, containing a CO₂ absorbent, inside the largerstorage bag but this method is highly dependent on the ability of CO₂ topenetrate the small bag to reach the absorbent and further, these smallbags are readily susceptible to a mucous-like build-up on their surfacewhich further cuts down on the transmission of CO₂ to the containedabsorbent.

Moreover, the small bags containing the CO₂ absorbent have thepossibility of highly contaminating the whole blood if they leak or areruptured in some manner.

Further, such a bag-within-a-bag design causes complicated fabricationproblems for the bag manufacturer.

SUMMARY OF PRESENT INVENTION

The present invention is directed to overcoming some of the problemsassociated with blood storage.

It is an object of the present invention to provide a method ofprolonging the period of storage of blood.

It is another object of the present invention to prolong the storage ofblood yet provide for wholesome blood that can be reinfused.

It is yet another object of this invention to provide a compositionwhich can be used in a whole blood storage bag to prolong the life ofthe whole blood stored therein.

It is still another object of this invention to provide a method ofeasier fabrication of a blood storage bag containing compositions whichwill prolong the life of the blood stored therein.

It is a further object of this invention to provide a blood storage bagwhich can prolong the life of whole blood and which can contributelittle or no whole blood contamination from the inventive composition.

There is, therefore, provided by this invention a composition of matterto be used in blood storage bags to inhibit the degeneration of theblood stored therein.

Such a composition consists of a curable silicone rubber containingcompounded therein essentially pure Ca(OH)₂ as a CO₂ absorbent.

For purposes of this invention, "essentially pure" means that theCa(OH)₂ useful in the invention contains at least 95% of Ca(OH)₂ asopposed to CaO. Further, "essentially pure" also preferably refers tothe highest grade of Ca(OH)₂ commercially available wherein the otherimpurities present are non-calcium materials and are present in veryminute quantities, i.e., on the order of parts per million.

There is also provided a method of storing blood and a blood storagesystem aimed at retarding degeneration of blood which consists ofequipping a blood storage bag with the above mentioned compounded rubberin the form of sheets, ribbons, kernels, granules, lumps or smallblocks. By equipping is meant that the compounded silicone rubber isadded to a blood storage bag along with the normal artificial medias.Such compounded silicone rubber thus acts as a CO₂ sink and therebyfacilitates the inhibition of the degeneration of the whole blood storedtherein.

DETAILED DESCRIPTION

The present invention contemplates contacting blood with cured siliconerubber containing essentially pure Ca(OH)₂ while the blood is stored. Itshould be understood for purposes of this invention, that because thecompounded rubber acts as a CO₂ sink, the surface area of the siliconerubber is an important factor in the selection of the physical form ofthe rubber to be used. To provide the necessary surface area the rubberis in the form of sheets, ribbons, kernels, granules, lumps or smallblocks, but some degree of care is necessary in choice ofconfigurations. For example, if granules are used to provide a largesurface area for absorption of large quantities of CO₂, care must betaken that the granules are not so small as to clog the infusion tubesand etc., of the storage bag. On the other hand, very large blocks orspheres of such rubber do not allow the requisite surface area forenough CO₂ absorption. Thus, design of the silicone pieces actually usedis dependent on the amount of CO₂ absorption that is actually desiredand this has to be balanced against possible mechanical malfunctionssuch as the clogging described above.

The silicone rubbers useful in this invention are those silicone rubberswhich are easily curable. Such methods of curing are preferably peroxidecures or platinum catalyzed curing of .tbd.SiH containing siloxanes with.tbd.Si alkenyl containing siloxanes.

Such silicone rubbers are well known in the art and further elaborationis not required herein.

The Ca(OH)₂ useful in this invention is Ca(OH)₂ that is essentiallypure. For obvious reasons, poorer grades of Ca(OH)₂ should not be usedbecause of the possible contamination problems. Preferably, Ca(OH)₂ isused that has greater than 95% Ca(OH)₂ as opposed to CaO. Further,preferably there should be used a Ca(OH)₂ that is the purest that can bereasonably obtained and which has impurities other than calcium in theparts per million range.

The Ca(OH)₂ is compounded into the uncured silicone rubber by any meansnormally used in the art such as a two-roll mill.

The largest amount of Ca(OH)₂ that can be used is limited by the amountthat can be milled into silicone rubber without detracting from the cureor the physical properties of the silicone rubber. The smallest amountis determined by the practical effects of absorbing power and quantitiesof CO₂ that are required to be absorbed. Usually, the amount ranges from5-30 parts based on 100 parts of the silicone rubber. The compoundedrubber is then cured by any convenient means which is conventional inthe art and which is dependent on the cure system used and the catalystrequired.

The uncured rubber can be molded to the proper shape or can be molded inblocks or sheets and cut or carved to the proper physical form.

At the present time, the preferred form both from a functional surfacearea and from a physical malfunctions aspect, is a 3 cm×3 cm piece thatis 1 cm thick and which has the corners slightly rounded.

The pieces are simple inserted into a blood storage bag during thefabrication of the bag and are allowed to float free within the bag.

The silicone rubber pieces adapt very nicely to sterilizationtechniques.

Now so that those skilled in the art can more fully understand theinvention, the following example is submitted.

EXAMPLE

Silicone rubber into which 17% by weight of pure Ca(OH)₂ had beencompounded and which had been cured were fashioned into 3.0 cm×3.0 cm×1cm blocks.

One block was inserted into each blood collection bag for this exampleby opening the bottom of an empty 600 ml transfer pack (Fenwal PL 130plastic), inserting the block and then resealing the pack with a hotiron and autoclaving for 20 minutes at 121° C.

Four hundred and fifty ml of blood were collected from healthy adultdonors into 67.5 ml of ACD (NIH formula A). The blood was allowed tostand for 15-30 minutes at room temperature, and then was centrifuged at4° C. at 4500×g for five to ten minutes in a Sorvall RC-3 centrifugewith a swinging bucket rotor. The plasma and buffy coat were bothpressed off, and 200 ml of preservative solution (BAGPM) containing101.4 mM of sodium bicarbonate (NaHCO₃), 14.3 mM sodium carbonate (Na₂CO₃), 1 mM bisodium phosphate (Na₂ HPO₄), 1 mM adenine, 55 mM glucoseand 0.5% mannitol was added for each volume of plasma expressed. Aftergentle but thorough mixing, the red cell suspension was asepticallytransferred into the bag containing the blocks. The red cells were thenstored at 4° C. for 42 days, and agitated for one minute either once atthe time of sampling, or five times weekly to ensure thorough mixing.

Erythrocyte ATP was quantitated by the hexokinase method (set out inBeutler, E., "Red Cell Metabolism", A Manual of Biochemical Methods,Second Edition, New York, 1975, Greene and Stratton), and theconcentration of 2,3-DPG was measured by a modification of the techniqueof Krimsky. (see Beutler, E. immediately above.) The pH was determinedby a technique which allowed anaerobic measurements at 0°-4° C.(Bensinger, T.A. Metro, J. and Beutler, E., redesigned apparatus foranaerobic measurement of blood pH at low temperatures, Amer., J. Clin.Path., 63, 264-268, 1975.)

Four units of blood were stored in BAGPM with the blocks and agitatedone time per week. The pH ranged from a high of 7.82±0.02 on day 7 to7.41±0.03 on day 42. Seven units containing the BAGPM and blocks wereagitated five times weekly and these units maintained their pH at7.86±0.03 on day zero to 7.55±0.08 on day 42.

Intracellular 2,3-DPG which was 13.16±0.88 μMole/g Hgb on day zero was10.22±2.35 μMole/g Hgb on day 42 in the units stored with BAGPM andblocks that were agitated five times weekly. 2,3DPG, which wasoriginally 15.3±1.6 μMole/g at the time of drawing, was 10.13±1.20μMole/g Hgb on day 42 if the system was agitated once weekly. In theunits agitated once weekly, the day 42 ATP was 2.40±0.64 μMole/g Hgb.The blood agitated five times weekly was 2.40±0.64 μMole/g Hgb. Theblood agitated five times weekly had ATP levels of 1.88±0.26 μMole/g Hgbafter 42 days of storage.

That which is claimed is:
 1. A composition useful for prolonging thestorage of blood which consists of a cured silicone rubber which hasbeen compounded in its uncured state with Ca(OH)₂.
 2. A composition asclaimed in claim 1 wherein the silicone rubber is present in a ratio of100 parts to 5-30 parts of Ca(OH)₂.
 3. A composition as claimed in claim1 wherein the silicone rubber containing the Ca(OH)₂ is cured by the useof peroxide catalysts and heat and the cure is effected through thereaction of unsaturated radicals on silicon in the silicone rubber.
 4. Acomposition as claimed in claim 1 wherein the silicone rubber containingthe Ca(OH)₂ is cured by the use of platinum catalysts and the cure iseffected through the reaction of hydrogen radicals on silicon andunsaturated radicals on silicon in the silicone rubber.
 5. A compositionas claimed in claim 1 contained in a blood storage bag in heterogenousadmixture with an artificial blood storage media.